Multimodal profiling of human postnatal thymocytes using CITE-seq

T cell development in the human thymus has primarily been studied using antibody-based approaches, such as flow cytometry, which can create difficulties in translating phenotypic findings to scRNA-seq data. In order to bridge this gap and obtain paired surface protein and RNA information for individual cells, we carried out CITE-seq with a 143-plex customised antibody panel on human postnatal thymocytes. This was further combined with TCR-seq for TRA and TRB loci to gain insights into the V(D)J recombination progress in developing cells. Using information from all three modalities, we annotated over 30 different stages of human T cell development, which align with known surface marker profiles. This data was utilised in the context of the human thymus spatial atlas for high-resolution spatial mapping of developing T cells, which revealed differences in the migration and maturation kinetics of CD4 and CD8 lineage single positive thymocytes. Thymocytes from 5 paediatric donors were stained with a TotalSeq-C panel containing antibodies against 143 surface markers and 7 isotype controls. Cells were sorted into CD3+ and CD3- subsets to enrich for immature stages and profiled using the 10X Genomics 5’ kit v2.